More than a decade ago a non-coding 22-nucleotide (nt) RNA (lin-4) was discovered that played an important role in the developmental timing of Caenorhabditis elegans. It was not realized, however, until just a just few years ago that small RNA molecules such as lin-4 are ubiquitous and play important regulatory roles in virtually all eukaryotes. Recent work has shown that prokaryotes and viruses also express small regulatory RNA molecules. Thus, in addition to large RNA molecules, such as messenger RNA (mRNA) and ribosomal RNA (rRNA), cells express an array of small RNA molecules, including 5.8S rRNA, 5S rRNA, transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA); micro RNA (miRNA), small interfering RNA (siRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), small temporary RNA (stRNA), tiny non-coding RNA (tncRNA), small scan RNA (snRNA), and small modulatory RNA (smRNA). Micro RNA molecules, which are processed from larger primary transcripts and range from 20-23 nucleotides in length, have emerged as a hot topic in molecular biology research because of their important roles in a wide range of biological processes, including gene regulation, cell differentiation, growth, and development, as well as certain disease states. Other small RNA molecules, such as siRNAs, are also involved in gene silencing and genome modification.
The long delay to the realization of the existence and importance of small RNA could, in part, be attributed to the fact that small RNA molecules are often unintentionally eliminated because of their small sizes from preparations of natural RNA populations. Furthermore, small RNA molecules represent a very small fraction in terms of weight of the total RNA population, and without removal of abundant RNAs and enrichment of small RNAs, their detection could be severely hampered. Historically, variations of two methods have been used to isolate RNA from biological samples. The first method relies on chemical extraction with organic solvents such as phenol and chloroform under acidic conditions to separate DNA and other biomolecules from the RNA, which is then concentrated by alcohol precipitation. Alcohol precipitation, however, does not quantitatively recover small RNA molecules. The second method relies on immobilization of RNA on a solid support binding matrix, such as silica. For this, the RNA-containing sample is mixed with a high salt solution or a salt and alcohol mixture to decrease the affinity of RNA for water and increase its affinity for the silica matrix. Small RNA, however, binds poorly to the support matrix under the conditions routinely used. Thus, most existing RNA preparation methods and commercial RNA purification kits are deficient in capturing small RNA.
With the recent surge of interest in miRNA and other small RNA molecules, the standard isolation procedures have been modified to facilitate the isolation of small RNA. These methods largely rely on phenol and chloroform extraction and step-wise alcohol fractionation. For example, U.S. Publication No. 2005/0059024 discloses a method in which a cell lysate is extracted with phenol and chloroform to partition the genomic DNA into an interphase between an organic lower phase and an aqueous upper phase. The aqueous upper phase is collected and mixed with a low percentage of alcohol and applied to a first binding matrix. The large RNA is immobilized onto the first matrix and the small RNA flow through the matrix. The flow-through fraction is then mixed with a higher percentage of alcohol and applied to a second binding matrix, to which the small RNA binds and can be recovered. Thus, small RNA can be isolated and purified using a multi-step procedure. A major drawback of the current methodology is the use of phenol and chloroform, not only because they pose potential health hazards but also because they are ineffective with certain biological material, such as plant tissues that are rich in phenolic or polyphenolic compounds. Another drawback of the current methodology is that phase separation and alcohol fractionation are laborious and time consuming, making them incompatible with high throughput and automation demands.
The present invention provides methods and compositions for the rapid isolation of small RNA from a variety of biological sources without using phenol and chloroform extraction or alcohol gradient fractionation.